The present invention relates to a convenient method for measuring a thiol group-containing compound and a thin membrane used for said method.
Thiol group-containing compounds have various functions attributable to reactivity of thiol groups. Accordingly, they are utilized in chemical industries, and they also play important roles in a living body such as glutathione, coenzyme A and so forth. For detection and quantitative assay of trace amounts of thiol group-containing compounds, methods having been applied so far include methods of direct measurement using separation and analysis means such as gas chromatography and liquid chromatography, or methods comprising the steps of reaction with a compound reactive to a thiol group such as maleimide derivatives, and then modification with a readily detectable compound such as a fluorescent dye and detection of the resulting compound. However, these methods have problems in that they require large-scale apparatuses or need skills for operation. Therefore, it has been desired to develop a method for easily and accurately measuring thiol group-containing compounds contained is biosamples in chemical laboratories, test facilities, clinical situations and so forth.
An object of the present invention is to provide a method for conveniently and accurately measuring a thiol group-containing compound. More specifically, the object of the present invention is to provide a method for conveniently and accurately measuring a thiol group-containing compound, for example, alkylmercaptans, arylmercaptans, amino acid derivatives such as cysteine and glutathione, proteins containing free thiol groups and so forth. Another object of the present invention is to provide a method for measuring a thiol group-containing compound which has the aforementioned characteristic features and is capable of accurately determining intratissular localization of said compounds which are derived from cancer cells localized in test tissues. A further object of the present invention is to provide a thin membrane used for the aforementioned method for measurement of a thiol group-containing compound.
The inventors of the present invention conducted researches to achieve the foregoing objects, and as a result, they found that, when a solution or a living tissue slice which contains a thiol group-containing compound was closely contacted with a thin membrane comprising micropaiticles of a substance selected from the group consisting of a metal and a metal compound together with a hydrophilic binder, the thiol group-containing compound contained in the solution or the living biological tissue slice interacted with the substance selected from the group consisting of a metal and a metal compound to cause color change such as change of color tone and coloration, and that the thiol group-containing compound contained in the sample was conveniently and accurately measurable by detecting the color change through visual observation, under optical microscope, or by spectroscopic means or the like. The inventors also found that detection ability of the aforementioned thin membrane was further improved by adding a crosslinking agent to the aforementioned thin membrane. The present invention was achieved on the basis of these findings.
The present invention thus provides a method for measuring a thiol group-containing compound, which comprises the steps of: (1) contacting a sample containing a thiol group-containing compound with a thin membrane comprising a microparticle of a substance selected from the group consisting of a metal and a metal compound and comprising a hydrophilic binder, and (2) detecting a color change on the thin membrane resulting from interaction of the thiol group-containing compound and the microparticle.
The following methods are examples of preferred embodiments of the aforementioned method of the present invention.
1 A method for measuring a thiol group-containing compound which comprises the steps of:
(1) dropping a solution containing a thiol group-containing compound onto a thin membrane comprising a microparticle of a substance selected from the group consisting of a metal and a metal compound and comprising a hydrophilic binder,
(2) optionally drying the dropped solution, and then subjecting the thiol group-containing compound in the solution to interact with the microparticle, and
(3) detecting a color change on the thin membrane resulting from the interaction of the thiol group-containing compound and the microparticle.
In the aforementioned embodiment, the step (2) is usually performed by leaving the thin membrane under saturated humidity at a temperature of from room temperature to 70xc2x0 C. for several minutes to several hours.
2 A method for measuring a thiol group-containing compound which comprises the steps of:
(1) contacting a frozen slice of a biosample with a thin membrane comprising a microparticle of a substance selected from the group consisting of a metal and a metal compound and comprising a hydrophilic binder and a crosslinking agent,
(2) subjecting the thiol group-containing compound in the biosample to interact with the microparticle, and
(3) detecting a color change on the thin membrane resulting from the interaction of the thiol group-containing compound and the microparticle.
In the aforementioned embodiment, the step (2) is usually performed by leaving the thin membrane, on which the slice is placed, under saturated humidity at a temperature of from room temperature to 70xc2x0 C. for several minutes to several hours.
3 A method for measuring a thiol group-containing compound which comprises the steps of:
(1) subjecting a solution containing a thiol group-containing compound to be absorbed into an absorptive medium,
(2) contacting the medium containing the absorbed solution with a thin membrane comprising a microparticle of a substance selected from the group consisting of a metal and a metal compound and comprising a hydrophilic binder,
(3) subjecting the thiol group-containing compound in the solution to interact with the microparticle, and
(4) detecting a color change on the thin membrane resulting from the interaction of the thiol group-containing compound and the microparticle.
In the aforementioned embodiment, the absorptive medium used in the step (1) may be, for example, paper, microfilter, gelatin film or the like, and the step (3) is usually performed by leaving the thin membrane under saturated humidity at a temperature of from room temperature to 70xc2x0 C. for several minutes to several hours.
As a preferred embodiment of the aforementioned method of the present invention, there is provided the aforementioned method wherein the thiol group-containing compound is selected from the group consisting of alkylthiols, arylthiols, amino acids and derivatives thereof, peptide compounds, and proteins. As a preferred embodiment of the aforementioned method of the present invention, there is provided the aforementioned method wherein the biosample is a sample isolated or collected from a mammal including human, more preferably, blood, blood plasma, blood serum, cancer tissue slice, gingival crevice exudate, destructive morbid tissue slice, or destructive morbid tissue extract (for example, rheumatic morbid tissue extract, alveolus blennorrhoea tissue extract and the like). There are also provided the aforementioned method wherein the metal or a metal that constitutes the metal compound is selected from the group consisting of metals of the 2nd period, 3rd period, 4th period, 5th period, and 6th period of the periodic table of elements; and the aforementioned method wherein the metal or a metal that constitutes the metal compound is selected from the group consisting of metals of Group VIb, Group VIIb, Group VIII, Group Ib, Group IIb, Group VIa and Group VIIa in the periodic table of elements.
According to another aspect of the present invention, there is provided a thin membrane used for measurement of a thiol group-containing compound, which contains microparticles of a substance selected from the group consisting of a metal and a metal compound, and hydrophilic colloids. According to a preferred embodiment of the thin membrane of the present invention, the aforementioned thin membrane which further contains a crosslinking agent is provided. This thin membrane is characterized by development of color change resulting from interaction of the thiol group-containing compound and the substance selected from the group consisting of a metal and a metal compound. As preferred embodiments thereof, there are provided the thin membranes for measurement of a thiol group-containing compound which are defined in the aforementioned methods {circle around (1)} through {circle around (3)}. According to further preferred embodiments of said thin membranes, there are provided the aforementioned thin membranes wherein the thin membranes are formed on a flat surface of a support such as object glass and polyethylen terephthalate film; and the thin membranes wherein an undercoat layer is provided between the thin membrane and the support.